bioRxiv preprint doi: https://doi.org/10.1101/252700; this version posted February 18, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Temporal and spatial factors that influence magnetotaxis in C. elegans Vidal-Gadea A.G.1,*, Caldart, C.S.2, Bainbridge, C.1, Clites, B.L.4, Palacios, B.4, Bakhtiari, L.A. 3, Gordon, V.D.3, Golombek D.A.2, and Pierce J.T.4,* 1School of Biological Sciences, Illinois State University, Normal, IL, USA 2Department of Science and Technology, National University of Quilmes, Argentina 3Department of Physics, University of Texas at Austin, Austin, TX, USA 4Department of 4Department of Neuroscience, University of Texas at Austin, Austin, TX, USA *corresponding authors: Andrés Vidal-Gadea ([email protected]); Jonathan Pierce ([email protected]) ABSTRACT Many animals can orient using the earth’s magnetic field. In a recent study, we performed three distinct behavioral assays providing evidence that the nematode Caenorhabditis elegans orients to earth-strength magnetic fields (Vidal-Gadea et al., 2015). In addition to these behavioral assays, we found that magnetic orientation in C. elegans depends on the AFD sensory neurons and conducted subsequent physiological experiments showing that AFD neurons respond to earth-strength magnetic fields. A new behavioral study by Landler et al. (2017) suggested that C. elegans does not orient to magnetic fields and raises issues that cast doubt on our study. Here we reanalyze Lander et al.’s data to show how they appear to have missed observing positive results, and we highlight differences in experimental methods and interpretations that may explain our different results and conclusions. Moreover, we present new data from our labs together with replication by an independent lab to show how temporal and spatial factors influence the unique spatiotemporal trajectory that worms make during magnetotaxis. Together, these findings provide guidance on how to achieve robust magnetotaxis and reinforce our original finding that C. elegans is a suitable model system to study magnetoreception. INTRODUCTION Most research on the neuronal basis for magnetosensation has focused on animals that migrate long distances by in part using the Earth’s magnetic field as a cue (Johnsen and Lohmann, 2005; Guerra et al., 2014). Although migrations by birds, butterflies, and turtles are magnificent in their own right, elucidating the cellular and molecular bases for magnetosensation is challenging in these complex animals. Favoring a simpler animal, we recently asked whether the nematode Caenorhabditis elegans was capable of magnetic orientation (Vidal-Gadea et al., 2015). C. elegans has proven historically important for the discovery of molecules used to sense odors, mechanical force, osmolarity, and humidity (Sengupta et al. 1996; O'Hagan et al., 2005; Colbert et al., 1997; Russell et al., 2014). Notably, each of these molecules share conserved functions in higher animals (Tobin & Bargmann, 2004; Arnadóttir & Chalfie, 1 bioRxiv preprint doi: https://doi.org/10.1101/252700; this version posted February 18, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 2010; Filingeri, 2015). If C. elegans displayed magnetoreception, potentially conserved molecular bases for this sensory modality may be studied using similar approaches. Using three distinct behavioral assays, we discovered that this tiny worm could orient its movement to artificial magnets or to the earth’s magnetic field (Vidal-Gadea et al., 2015). By leveraging genetic tools specific to C. elegans, we next determined that the AFD sensory neurons were required for magnetic orientation and began to uncover signal transduction components required for magnetosensation (Vidal-Gadea et al., 2015). We also found that similar to magnetotactic bacteria, wild C. elegans strains isolated from around the world oriented to magnetic fields in a manner reflecting the magnitude and direction of the geomagnetic field at their point of isolation (Blakemore, 1975; Vidal-Gadea et al., 2015). Our behavioral and physiological results provide strong evidence that C. elegans senses and orients to magnetic fields. Recently, Landler et al. (2017) performed additional sets of behavioral experiments to confirm whether C. elegans orients to magnetic fields. They reported negative results for all three experiments and conclude that C. elegans is not a suitable model system to study the molecular basis for magnetoreception. On first inspection, the experiments done by Landler et al. (2017) resemble those from our study with additional levels of control. We discuss subtle but important differences in experimental methods, controls, and execution that might have contributed to their negative results. We also discuss how behavioral results obtained by independent labs are consistent with our original findings. To help groups get started with C. elegans magnetotaxis, we present a new spatiotemporal analysis of worms orienting to magnetic fields along with suggestions on how to simplify our assays and control factors to ensure more robust results. Lastly, Landler et al. (2017) discuss conceptual issues with our findings and interpretations. First, they suggest that directional information is absent in the magnetic field used in our magnetotaxis assay. Second, they suggest that a tentative explanatory hypothesis that we put forward– that C. elegans strains isolated from different locations on the globe may migrate at a specific angle to the magnetic field, perhaps as a way to orient optimally up or downwards when burrowing - is infeasible. We address these two issues by showing data from experiments demonstrating that the magnetic field does provide directional information in our magnetotaxis assay. Furthermore, thanks to this challenge, and the new experiments it prompted, we are now able to reconcile the results from our uniform and radial magnetic field experiments. We finish by identifying plausible mechanisms for how worms may use the directional information provided by a magnetic field to migrate along a specific vector. Overall, the new data and updated interpretations offered in this response will likely help readers understand the discrepancy between the results reported in Landler et al. (2017) and those reported by our three groups. With this additional information, we hope that more researchers will be drawn to study the cellular molecular basis for magnetoreception using C. elegans. 2 bioRxiv preprint doi: https://doi.org/10.1101/252700; this version posted February 18, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. RESULTS AND DISCUSSION This response is divided into four sections. We begin by summarizing of our previous results. The second section will discuss overt and potential differences between the assays conducted by Vidal-Gadea et al. (2015) and Landler et al. (2017). Third, we provide additional experiments and results from an independent lab confirming our original findings. In the last section, we discuss conceptual issues related to trajectories that worms make during magnetic orientation. Summary of previous results Our previous paper included a large number of experiments and controls ranging from cellular calcium recordings, neuronal ablations, and cell-specific gene rescues, mutant analyses, and behavioral studies. The present challenge restricts itself to our behavioral experiments. We developed and performed three distinct behavioral assays to test whether C. elegans orients to magnetic fields (Vidal-Gadea et al., 2015) summarized below. Burrowing assay: We tested whether British (N2) strain worms displayed a preference for burrowing up or down when injected into an agar-filled cylinder. We found that worms preferred to migrate down when starved (which we defined as 30 or more minutes away from food), and up when well-fed (which we defined as less than 30 minutes away from food). To test whether vertical bias was controlled by gravity or the magnetic field, we experimentally inverted an earth-strength magnetic field and found that the directions of migration were reversed. In Australia, the earth’s magnetic field has a polarity that is opposite that in Britain; we found that worms isolated from Australia migrated in the opposite direction to British worms. These results are consistent with the idea that the magnetic field, not gravity, provides the major cue for burrowing direction. Horizontal plate assay: We tested how worms migrate in a uniform magnetic field generated by a magnetic coil system across a 10-cm diameter assay plate. We found that British worms moved randomly when the earth’s magnetic field was cancelled by the coil system, but migrated at a single specific angle with respect to magnetic north in the presence of an earth-strength field directed across horizontally positioned assay plate. Analogous to our Burrowing Assay results, the angle that worms migrated with respect to the field depended on the satiation state of the worms